Research supported by this grant in recent years has focused on what we have defined as the non-heme iron reductive paradigm for combating oxidative and nitrosative stress. This novel paradigm involves reductive scavenging of toxic reduced oxygen and nitrogen species by a group of bacterial and archaeal non-heme iron enzymes. Genetic evidence associates the functions of these enzymes in numerous bacterial and archaeal species. Based on genome sequences, these enzymes are found predominantly in air-sensitive bacteria, including those that constitute the majority of the normal human gut flora as well as human pathogens. These enzymes may protect against the oxidative and nitrosative burst of macrophages, which is the human host's initial response to infection. In the present renewal proposal, one specific aim focuses on the mechanism of reductive nitric oxide scavenging by a novel enzyme containing a combination of flavin and diiron cofactors at its active site. Single turnover experiments will be used to identify the Fe-NO species formed upon reactions of the diiron sites with nitric oxide and to determine their catalytic relevance. These themes are then logically extended to bacterial O2 sensing and signaling, induction of biofilm formation and virulence, all involving bacterial non-heme iron proteins. Enzymes from Vibrio cholerae, the bacterium causing epidemic cholerae, catalyzing formation and decay of the bacterial second messenger, cyclic-di-guanosine monophosphate, are the focus in these latter two specific aims. These enzymes are widespread in bacteria but have not been found in higher eukaryotes. Inhibitors of these enzymes could, therefore, constitute new classes of antibiotics. Intelligent design of antibiotics requires an understanding of their catalytic and/or sensing/signaling mechanisms. These mechanisms are the overall goal of this research.